Caracterização bioquímica da Cutinase-Like de Cryptococcus flavus expressa em Komagataella phaffii para degradação de biopolímeros e aplicações biotecnológicas

Abstract

More than 350 million tons of plastic are produced annually in the world, of which 76% are disposed of incorrectly. Biopolymers are an alternative to this environmental problem. However, decades are required for complete degradation of biopolymers under ambient conditions. In this context, cutinases are able to catalyze the hydrolysis of polyester esters, having several biotechnological applications, among them, the hydrolysis of biopolymers such as PBS, PLA, PLC and others. The extracellular cutinase of Cryptococcus flavus I-11 (CfCLE) has important catalytic properties such as resistance to organic solvents and surfactants, which may enable the biotechnological application of this enzyme in the processing of biopolymers. The objective of the present work was to biochemically characterize the CfCLE enzyme, produced in a heterologous expression system of Komagataella phaffii, cloned under the control of the AOX1 promoter, in order to biotechnologically apply this enzyme. K. phaffii recombinants were generated and screened based on enzymatic activity and amount of proteins, in order to select the best transformant. Enzyme preparations with high purity and specific activity were obtained through scalable fermentation and purification processes. The results of biochemical characterization of recombinant CfCLE (CfCLEr) demonstrate that the protein has an optimal temperature of 30°C and thermo stability up to 50°C for 4 hours, an optimal pH of 8.5 and activity in the pH range 6-11, resistance to various organic solvents such as methanol and ethanol, resistance to surfactants such as Triton and Tweens, increased activity in the presence of divalent ions, greater specificity for 8-carbon esters and 4-carbon triglycerides. It was demonstrated in this work that the enzymatic preparations of purified CfCLEr are able to catalyze the hydrolysis of PET films and 84% polycaprolactone (PLC), 52% polylactic acid (PLA) and 32% polyhydroxybutyrate (PHB), which opens a perspective for industrial application in improving the degradation of biopolymers.